Recent studies suggested that regulatory T cells (Tr) expressing transcription factor Foxp3 play a key role in keeping in check autoreactive T cells escaping negative selection in the thymus and peripheral tolerance induction. During previous funding period, we found protective Tr cells themselves utilize TCR with a heightened reactivity towards self-peptide MHC class II complexes. The increased affinity TCR signals together with additional poorly understood signals are likely essential for the differentiation of Tr cells. Nevertheless, TCR specificity requirement for Tr mediated protection against autoimmunity and the contribution of distinct APC types to Tr generation in the thymus and their maintenance and function in the periphery remain unknown. Several types of thymic APC can contribute to differentiation of Tr cells and we hypothesize that the ability of the Tr population to protect against autoimmune inflammation in distinct tissues and organs is differentially shaped by particular types of thymic and peripheral MHC class II expressing APC. In the current proposal we will employ a unique set of genetic tools to test these hypotheses by investigating the suppressive potential of Tr cells selected by the thymic cortical epithelial cells alone or together with the thymic medullary epithelial cells or dendritic cells to prevent autoimmune lesions in various organs. We will also examine how the repertoire of TCR displayed by Tr cells is shaped by MHC class II molecules displayed by distinct types of APC. Finally, we will test a requirement for distinct types of APC for thymic differentiation and peripheral maintenance of Tr cells, expressing a single TCR, and examine the ability of these cells to prevent autoimmunity. Together, the studies described in the current application will provide novel insights into cellular mechanisms involved in differentiation and maintenance of a functional protective repertoire of regulatory T cells. PUBLIC HEALTH RELEVANCE: Regulatory T cells recently emerged as a key mechanism preventing autoimmunity and excessive tissue damage in the course of the immune response to viral, bacterial, and parasitic infections. Furthermore, these cells greatly diminish anti-tumor immunity. In studies described in this application, we will investigate the ways a functional protective repertoire of regulatory T cells is shaped during their differentiation. This work will lead to a better understanding of powerful protection against immune-inflicted damage of specific organs and tissues afforded by regulatory T cells and facilitate development of novel therapeutic approaches to treatment of autoimmune diseases.